This invention relates to diagnostic detection of viral nucleic acids, and specifically relates to compositions and assays for detecting HIV-1 sequences using transcription-mediated nucleic acid amplification and probe detection of amplified sequences.
Human immunodeficiency virus 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS) and AIDS related syndrome (ARC). Because the infectious virus is transmissible in body fluids, including blood and plasma, it is important to detect infected body fluids before antibodies to the virus are detectable or symptoms are evident in the infected individual. For protection of patients who might otherwise receive HIV-1-infected body fluid (e.g., whole blood or plasma during transfusion), or products derived from blood or plasma, it is particularly important to detect the presence of the virus in the body fluid to prevent its use in such procedures or in products. It is also important that procedures and reagents used in detecting HIV-1 be able to detect relatively low numbers of viral copies which may be present in an infected individual.
Assays and reagents for detecting HIV-1 have been previously disclosed in, for example, U.S. Pat. Nos. 5,008,182, 5,594,122, 5,688,637 and 5,843,638; European Patent Nos. EP 178 978 B1, EP 181,150 B1 and EP 185,444 B1; published European Patent Application Nos. EP 403,333, EP 462,627 and EP 806,484; and PCT No. WO 99/61666.
The present invention includes oligonucleotide sequences used as primers for amplification and probes for detection of HIV-1 nucleic acid present in a biological sample, using an assay that preferably includes transcription-mediated nucleic acid amplification (e.g., as previously disclosed by Kacian et al., U.S. Pat. Nos. 5,399,491 and 5,554,516). The preferred detection method uses known homogeneous detection techniques to detect, in a mixture, a labeled probe that is bound to an amplified nucleic acid (as disclosed, for example, in Arnold et al. Clin. Chem. 35:1588-1594 (1989); Nelson et al., U.S. Pat. No. 5,658,737; and Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). The present invention also includes nucleic acid oligonucleotide sequences that are useful for capturing the HIV-1 target using nucleic acid hybridization techniques that preferably use magnetic particles in separation of the captured target (Whitehead et al., U.S. Pat. Nos. 4,554,088 and 4,695,392).
According to one aspect of the invention, there are provided oligomers comprising a base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:57. One embodiment includes oligomers wherein the base sequence is that of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:45. Another embodiment includes oligomers further comprising a backbone that includes at least one 2xe2x80x2-methoxy RNA group, at least one 2xe2x80x2 fluoro-substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, at least one methylphosphonate linkage or any combination thereof. Another embodiment includes oligomers in which the base sequence comprises the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:45, and the backbone comprises at least one 2xe2x80x2-methoxy RNA group.
According to another aspect of the invention, there are oligomers consisting of a base sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56. In one embodiment, the oligomer has a base sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:16. In another embodiment, the base sequence of the oligomer is joined by a backbone that includes at least one 2xe2x80x2-methoxy RNA group, at least one 2xe2x80x2 fluoro-substituted RNA group, at least one peptide nucleic acid linkage, at least one phosphorothioate linkage, at least one methylphosphonate linkage or any combination thereof.
According to another aspect of the invention, there are provided labeled oligomers comprising a base sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56; and a detectable label joined directly or indirectly to the base sequence. In one embodiment, the detectable label is a luminescent compound. In another embodiment, the base sequence is joined by a backbone comprising at least one 2xe2x80x2-methoxy RNA group. One embodiment is a labeled oligomer having the base sequence of SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, and the label that is a chemiluminescent compound. A preferred embodiment is a labeled oligomer having the base sequence of SEQ ID NO:16 containing an inosine at residue 7, and an acridinium ester compound as the label.
According to another aspect of the invention, there is provided a method of detecting HIV-1 RNA in a biological sample, comprising the steps of: providing a biological sample containing HIV-1 RNA; contacting the biological sample with at least one capture oligomer comprising a base sequence that hybridizes specifically to a target region in LTR or pol sequences of HIV-1 RNA, thus forming a capture oligomer:HIV-1 RNA complex; separating the capture oligomer:HIV-1 RNA complex from the biological sample; then amplifying the LTR or pol sequences, or a cDNA made therefrom, using a nucleic acid polymerase in vitro to produce an amplified product; and detecting the amplified product using a labeled detection probe that hybridizes specifically with the amplified product. In one embodiment, the contacting step uses a capture oligomer that further comprises a tail sequence that binds to a complementary sequence immobilized on a solid support. In another embodiment, the base sequence of the capture oligomer that hybridizes specifically to a target region in LTR or pol sequences comprises a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:19 or SEQ ID NO:57. In another embodiment, the capture oligomer comprises the base sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20 or SEQ ID NO:45, or is any combination of oligomers of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20 or SEQ ID NO:45. In a preferred embodiment, the capture oligomer is any combination of at least two oligomers having base sequences selected from the group of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. In one embodiment, the capture oligomer is a combination of oligomers having base sequences of SEQ ID NO:20 and SEQ ID NO:6, or SEQ ID NO:45 and SEQ ID NO:6. In another embodiment, the amplifying step uses at least two amplification oligomers that bind specifically to LTR or pol sequences or complementary sequences thereof. Preferably, the amplifying step uses at least two amplification oligomers for amplifying LTR sequences selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38. Another embodiment uses, in the amplifying step, at least two amplification oligomers for amplifying pol sequences selected from the group consisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID.NO:14, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44. In another embodiment, the amplifying step comprises a transcription-associated amplification method that includes at least one promoter-primer comprising a promoter sequence that is recognized by an RNA polymerase when the promoter sequence is double stranded, wherein the promoter sequence is covalently attached to the 5xe2x80x2 end of a LTR-specific sequence selected from the group consisting of SEQ ID NQ:7, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NQ:29, SEQ ID NO:31 and SEQ ID NO:33, or a pol-specific sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14; and at least one primer comprising a LTR-specific sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or a pol-specific sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:42, provided that at least one LTR-specific promoter-primer is combined with at least one LTR-specific primer for amplifying a LTR target region, or at least one pol-specific promoter-primer is combined with at least one pol-specific primer for amplifying a pol target region. In one embodiment, the amplifying step comprises a transcription-associated amplification method that includes at least one promoter-primer having a LTR-specific sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:34, or a pol-specific sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NQ:15, SEQ ID NO:43 and SEQ ID NO:44; and at least one primer having a LTR-specific sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NQ:36, SEQ ID NO:37 and SEQ ID NO:38, or a pol-specific sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:42, provided that at least one LTR-specific promoter-primer is combined with at least one LTR-specific primer for amplifying a LTR target region, or at least one pol-specific promoter-primer is combined with at least one pol-specific primer for amplifying a pol target region. Preferably, the amplifying step uses any of the following combinations of promoter-primers and primers: promoter-primers of SEQ ID NO:13 and SEQ ID NO:15, with primers of SEQ ID NO:10 and SEQ ID NO:11; promoter-primers of SEQ ID NO:13 and SEQ ID NO:15, with primers of SEQ ID NO:42 and SEQ ID NO:11; promoter-primers of SEQ ID NO:43 and SEQ ID NO:15, with primers of SEQ ID NO:10 and SEQ ID NO:11; promoter-primers of SEQ ID NO:13 and SEQ ID NO:44, with primers of SEQ ID NO:10 and SEQ ID NO:11; promoter-primers of SEQ ID NO:7, SEQ ID NO:13 and SEQ ID NO:15, with primers of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; a promoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:9; a promoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:35; a promoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:36; a promoter-primer of SEQ ID NO:30, and a primer of SEQ ID NO:9; a promoter-primer of SEQ ID NO:30, and a primer of SEQ ID NO:36; a promoter-primer of SEQ ID NO:32, and a primer of SEQ ID NO:9; a promoter-primer of SEQ ID NO:34, and a primer of SEQ ID NO:36; a promoter-primer of SEQ ID NO:13, and a primer of SEQ ID NO:10; or a promoter-primer of SEQ ID NO:7, and a primer of SEQ ID NO:9. In one embodiment, the detecting step uses at least one labeled detection probe having a base sequence selected from the LTR-specific group consisting of SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, or the pol-specific group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, or a combination thereof. In another embodiment, the detecting step uses a combination of at least two labeled detection probes having the base sequences of SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18. Preferably, the labeled detection probe of SEQ ID NO:16 has an inosine at position 7. One embodiment, in the detecting step, uses at least one labeled detection probe having a base sequence selected from the LTR-specific group consisting of SEQ ID NO:16, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41. Another embodiment, in the detecting step, uses at least one labeled detection probe having a base sequence selected from the pol-specific group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56. In one embodiment, the detecting step uses at least one labeled detection probe that includes at least one 2xe2x80x2-methoxy backbone linkage. Another embodiment includes the contacting step that uses capture oligomers having the sequences of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; the amplifying step that uses promoter-primers having the sequences of SEQ ID NO:8, SEQ ID NO:13 and SEQ ID NO:15 and primers having the sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and the detecting step that uses labeled detection probes having the sequences of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. In one embodiment, the contacting step uses at least two capture oligomers that hybridize to different sequences in the target region; the amplifying step uses at least two different promoter-primers that hybridize to a first set of sequences within the target region and at least two different primers that hybridize to a second set of sequences within the target region; and the detecting step uses at least two labeled probes that bind specifically to different sequences located between the first set and second set of sequences within the target region. In another embodiment, the contacting step uses capture oligomers having the sequences of SEQ ID NO:4 and SEQ ID NO:6; the amplifying step uses promoter-primers having the sequences of SEQ ID NO:13 and SEQ ID NO:15 and primers having the sequences of SEQ ID NO:10 and SEQ ID NO:11; and the detecting step uses labeled probes having the sequences of SEQ ID NO:17 and SEQ ID NO:18. In a preferred embodiment, the amplifying step uses at least two promoter-primers that hybridize to a first set of overlapping sequences within the target region, at least two primers that hybridize to a second set of overlapping sequences within the target region, or a combination thereof.
According to another aspect of the invention these is provided a kit comprising a plurality of oligomers having the sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18, wherein the oligomers having the sequences of SEQ ID NO:17 and SEQ ID NO:18 are labeled with a detectable label. In one embodiment, the kit further includes oligomers having the sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:16, wherein the oligomer of SEQ ID NO:16 is labeled with a detectable label.